USP50 suppresses alternative RecQ helicase use and deleterious DNA2 activity during replication

Mammalian DNA replication relies on various DNA helicase and nuclease activities to ensure accurate genetic duplication, but how different helicase and nuclease activities are properly directed remains unclear. Here, we identify the ubiquitin-specific protease, USP50, as a chromatin-associated protein required to promote ongoing replication, fork restart, telomere maintenance, cellular survival following hydroxyurea or pyridostatin treatment, and suppression of DNA breaks near GC-rich sequences. We find that USP50 supports proper WRN-FEN1 localisation at or near stalled replication forks. Nascent DNA in cells lacking USP50 shows increased association of the DNA2 nuclease and RECQL4 and RECQL5 helicases and replication defects in cells lacking USP50, or FEN1 are driven by these proteins. Consequently, suppression of DNA2 or RECQL4/5 improves USP50-depleted cell resistance to agents inducing replicative stress and restores telomere stability. These data define an unexpected regulatory protein that promotes the balance of helicase and nuclease use at ongoing and stalled replication forks.

Supplementary Figure 1.Alignment of USP50 protein sequences from various species.Ile-141 in human USP50 (Q70EL3) is highlighted in yellow.

Supplementary Figure 2.
A. Amylose-bead precipitation alone (beads only), or precipitation of MBP (Empty), or MBP-USP50 fusion generated in bacteria and mixed with Ubiquitin (unconjugated).10% of the Ub input was loaded in the lane on the left.B. Amylose-bead precipitation alone (beads only), or precipitation of MBP only (Empty), or MBP-USP50 fusion generated in bacteria and mixed with K48-linked ubiquitin chains.10% of the Ub input was loaded in the lane on the left.
A. Immunoblot of two Hela cell lines transiently expressing wild-type FLAG-USP50, with and without IPTG to induce a non-targeting control sequence or shUSP50.
B. Immunoblot of Dox-inducible, shRNA-resistant FLAG-USP50 wild-type at the protein level (WT) or I141R HeLa cell lines treated, or not, with Dox.All cells were derived from cell line 2 (shown in A) and treated with IPTG to induce shUSP50.
C Diagram to illustrate structures identified in the DNA fibre assay to measure fork kinetics after sequential CldU and IdU labelling.
D HeLa cells, treated with siNTC (-) or shUSP50 (+), were incubated sequentially with CldU and IdU before 3 hours 5mM HU added.Tract lengths for each label were measured and the ratios determined.n=3, >200 fibres per condition.

G
. Top: illustration of the detection of PAR using the poly-ADP-ribose binding reagent MABE1031.Bottom: quantification of mean fluorescence intensities of PAR in cells ±shUSP50, ±FLAG-USP50, ±10 µM PARGi (for the last 20 mins).The mean intensity of PAR was normalised to the mean fluorescent intensity of S phase PAR in the presence of PARGi.n=3.H. Representative ScanR images for G. I. Top: illustration of the detection of PAR using the poly-ADP-ribose binding reagent MABE1031.Bottom: quantification of mean fluorescence intensities of PAR in cells ±shUSP50, ±FLAG-USP50, ±10 µM PARGi (for the last 20 mins).siNTC cells were incubated with 10 µM FEN1i (positive control) or 10 µM PARPi (negative control).n=1.J. Representative ScanR images for I. K. Colony survival of HCT116 (left) and RKO (right) cells treated with siNTC or siUSP50 ± siWRN.n=3.L. Mean % of chromatids with lagging strand telomere loss after shUSP50 and complementation with WT or I141R FLAG-USP50.Representative CO-FISH image is shown (right) n=3, scored chromatid numbers per experiment: NTC Where included, graphs indicate the mean ± SEM, exact P values are shown, and number of biological repeats is listed (n).All statistical analysis in this figure was performed using a two-tailed unpaired t-test.Source data are provided with this paper.A.Western blot analysis of HeLa cells treated with siNTC or siWRN, blots are probed forWRN and  Vinculin (loading control).Blots probed for WRN and α-tubulin (loading control).D.The % of stalled forks from GFP-WRN HeLa variants, WT-WRN, KM, or EA induced by Dox and treated with siNTC (-) or siUSP50 (+).n=3, >200 fibres per condition.E.53BP1 foci per EdU positive cell in WT-WRN, KM, or EA expressing cells on a background of siNTC (-) or siUSP50 HeLa cells (+), treated with 5 mM HU for 3 hours.n=3,>120 cells per condition.F.Left: western blot of siRNA resistant Myc-FEN variants; WT-FEN1, and E395K-FEN1 (EK).Blots were probed for Myc and vinculin (loading control).Middle: western blot of siNTC or shUSP50 expressing cells.Blots were probed for FEN1 and vinculin.Right: western blot of siNTC or siFEN1 treated cells.Blots were probed for FEN1 and Vinculin.